PROJECT SUMMARY Vibrio cholerae causes an acute diarrheal disease that is estimated to lead to 3 to 5 million cases of cholera and causes over 100,000 deaths annually. The increasing burden of cholera, the inability to achieve benchmarks for sanitation and safe water, and the emergence of more virulent strains of V. cholerae suggest that more aggressive approaches to preventing cholera, including vaccination programs, are needed. However, currently available killed whole cell vaccines generate immunity that rapidly wanes and provides only partial protection; especially in young children. In contrast, natural infection with V. cholerae induces 90-100 % protection against re-infection that lasts for up to 10 years in adults and children. It remains unknown why current vaccines are markedly less effective than natural infection. Understanding mucosal immunity and mechanisms regulating homing of immune cells to mucosal tissues in humans is of key importance, not only for V. cholerae infection, but for many other pathogens with a mucosal route of infection. A novel live attenuated cholera vaccine (Vaxchora) was recently approved in the US for use in travelers. This vaccine has showed excellent protection in human challenge studies. However, little is known about the long-term duration of protection, what determines the longevity of cholera specific memory B cells and mucosal plasmacells, or what governs plasmacell migration to, and survival within, the intestinal mucosa. In the current proposal, we seek to identify key differences in the early B cell responses after vaccination with the live attenuated vaccine, that may predict long-term humoral immunity. We propose to draw upon existing collaborations between the Emory Vaccine Center, the Emory Hope Clinic and the Massachusetts General Hospital/Harvard Medical School (MGH/HMS) to address this question in humans. Specifically, we will study both early and long term B cell responses in peripheral blood and in intestinal mucosae samples, using both global and single cell approaches. These studies will provide unprecedented insight into heterogeneity of the acute plasmablasts responses to cholera, their origin and activation process, affinity maturation and class switching, mucosal homing potential, ability to provide long-lived immunity after infection is resolved, and the antigenic specificity of the BCR, at a single cell level. Finally, imaging experiments will provide mechanistic insight into the mode of action of anti-cholera antibodies. These studies will generate a large number of monoclonal antibodies against cholera that might have both diagnostic and therapeutic uses. Findings herein may also instruct future vaccine development for this important human pathogen.